PROJECT SUMMARY/ABSTRACT The p53 tumor suppressor protein regulates critical control points of cellular homeostasis, including protecting cellular DNA from damage that could predispose to cancer. Indeed, a fully operational p53 signaling network is required for the pro-apoptotic activity of many chemotherapeutic agents. Whereas p53 can be mutated or deleted in cancer to avoid cell cycle arrest or apoptosis, a frequent alternative mode of p53 suppression relies on overexpression of the negative regulators HDMX and HDM2, which neutralize p53 through protein interaction. The individual contributions of HDMX and HDM2 to p53 suppression and chemoresistance in human cancers are largely unknown. Interestingly, we observe a relatively consistent level of HDM2 expression across a large and diverse panel of cancer cells, whereas HDMX manifests more variable expression, with subtypes such as acute myeloid leukemia exhibiting among the highest HDMX levels. What's more, HDMX scored in the top gene dependencies for several AML cell lines that also maintain wild-type p53 expression. These findings suggest that selective targeting of HDMX could both inform the mechanistic role of HDMX dependency in cancer and provide a therapeutic strategy for restoring the p53-pathway in HDMX-driven cancers. Whereas small molecule and stapled peptide inhibitors, which respectively inhibit HDM2 selectively or target both HDM2 and HDMX, have now been advanced to clinical testing, no validated HDMX-specific agent has been developed or validated to date. In addition, the influence of ligand engagement of HDMX and HDM2 on protein structure and the conformational dynamics of their p53 complexes is essentially unknown. Here, I aim to apply chemical, structural, cellular, and in vivo approaches to identify the binding determinants for selective targeting of HDMX, evaluate the structural and functional consequences of selective HDMX engagement, and advance a therapeutic strategy for reactivating p53-mediated apoptosis in HDMX-driven chemoresistant cancers. To achieve these goals, I propose three experimental aims: (1) synthesize libraries of structurally-reinforced alpha-helices modeled after the p53 transactivation domain to characterize their interactions with HDMX and HDM2, and identify selectivity determinants, (2) elucidate the conformational effects of SAH-p53 peptide engagement on HDMX and HDM2 proteins, alone and in complex with p53, and (3) advance lead HDMX-specific peptide inhibitors to cellular and in vivo testing to validate specific inhibition of HDMX as a therapeutic strategy for overcoming chemoresistance in cancers that retain wildtype p53 expression. I am eager to embark on a rigorous and multidisciplinary training program to accomplish the above-proposed graduate studies at Harvard Medical School and the Dana-Farber Cancer Institute, and look forward to an impactful physician-scientist career at the interface of chemistry, cancer biology, experimental therapeutics, and clinical oncology.